Protozoan parasites cause diseases such as malaria, trypanosomiasis, Chagas"" disease, leishmaniasis, giardiasis, and amoebiasis. These and other parasitic diseases historically have occurred in tropical and sub-tropical areas where they cause widespread suffering of human populations. Although they receive little attention in the Western world, protozoan diseases affect more people worldwide than diseases brought on by any other biological cause (Heyneman, 1988).
Today, malaria remains the most destructive single infectious disease in the developing world. It is responsible for more human death, energy loss, more debilitation, more loss of work capacity, and more economic damage than any other human ailment facing the world today (Heyneman, 1988). The World Health Organization estimates that 1 to 2 million deaths are caused by malaria each year in Africa alone; most of these are children under the age of five (World Health Organization, 1991). In addition, over 300 million people worldwide are believed to be chronically infected, and each year nearly one third of these individuals will suffer acute manifestations of the disease.
Today, the pathologic capacity of protozoa is being increasingly demonstrated in the Western world among AIDS (Acquired Immunodeficiency Syndrome) victims. AIDS depletes the immune system of affected individuals. This allows opportunistic agents to infect AIDS patients, which agents otherwise would be defeated by an active immune system. Several protozoa have proved especially opportunistically infectious in AIDS patients, including Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Pneumocystis carinii (which may be a fungal or protozoal pathogen), and Toxoplasmosis gondii. 
Despite the prevalence and significance of protozoan infections, therapy for these diseases is generally poor or in need of improvement. Many chemotherapeutic agents used to treat protozoan infections are non-specific cytotoxins that are highly toxic and cause severe side effects in patients. However, these drugs are used because there are no better alternatives. For example, giardiasis and amoebiasis are treated using metronidazole (a nitroimidazole), but the mutagenic potential of this drug (Campbell, 1986) and its adverse interaction with alcohol are problematic. For trypanosomiasis and leishmaniasis standard therapies (suramin, melarsoprol, and pentavalent antimonials) are dangerously toxic, occasionally fatal, and often ineffective (Mebrahtu, 1989; Grogl et al., 1992). Other drugs are becoming ineffective due to emerging resistance. In the case of malaria, effective therapy previously has been provided by chloroquine but its efficacy is now threatened by the rapid emergence of drug resistant strains of Plasmodium falciparum, the causative agent for the most severe, often fatal, form of the disease (Cowman, 1990). Other protozoal infections such as cryptosporidiosis or Chagas"" disease have no proven curative agent.
New therapeutic agents have been developed to treat protozoan infections. For example, Winter et al., U.S. Pat. No. 5,977,077, which is incorporated herein by reference, describes certain xanthone analogs which have sub-10 xcexcM IC50s (some having sub-1xcexcM IC50s), against Plasmodium and Leishmania. Despite these new xanthone analogs useful for treating infectious diseases, particularly protozoan diseases, there still is a need for new agents with comparable or better activities and reduced undesirable attributes, such as toxicity. A diverse array of therapeutic agents also is desireable to prevent or reduce the development of drug-resistant protozoan strains.
The present invention concerns new compounds which are useful, amongst other things, as antiparasitic agents. Methods for using these new compounds and certain known compounds as anti-parasitic agents are described. These antiparasitic agents form complexes with heme and with porphyrins with superior affinity and are therefore useful in a variety of other applications. The invention also is directed to compounds with broad-spectrum anti-microbial activity.
As a result of studies aimed at developing new anti-parasitic agents, the present inventors have discovered that xanthones and a wide range of xanthone derivatives and structurally related compounds, as represented by Formula X below, have potent anti-parasitic activity. The compounds have broad-spectrum anti-microbial activity, including anti-fungal activity against Candida albicans and Aspergillus fumigatus, and may possess antiviral activity as well.
Formula X 
With reference to Formula X, A is oxygen, substituted antimony (stibium), sulfur or N-R"" where Rxe2x80x2 is H, OH, alkyl, haloalkyl, aryl or haloaryl. Examples of substituted antimony groups include antimonial oxides and antimony substituted with hydroxy, chlorine, alkyl and aryl groups (e.g. SbCl, SbCl3, SbOH, Sb(O)(OH)). R1-R8 are independently selected from the group consisting of H, OH, halogen, aryl, arylamine, alkyl, alkene, substituted alkyl (such as alkylamine, alkylthio, haloalkyl, and substituted alkyls having two or more of such substituents), alkoxy, particularly lower alkoxy, such as methoxy, substituted alkoxy (such as alkoxylamine, cycloaminoalkoxy, dialkylaminoalkoxy, such as diethylaminoethoxy, haloalkoxy, and alkoxy groups having two or more of such substituents) amino, ester, ether, nitro groups and O-linked and C-linked carbohydrates. Alkyl and alkoxyl groups often include 10 or fewer carbon atoms in a straight or branched chain, and are referred to as xe2x80x9clowerxe2x80x9d alkyl or alkoxy groups. Y is selected from the group consisting of NO, NOH, Cxe2x95x90O, CHxe2x80x94OH, Sxe2x95x90O, and SO2.
Compounds having Y=carbonyl further satisfy Formula X1, where A and R1-R8 are as stated above with reference to Formula X.
Formula X1 
Certain Formula X1 compounds are compounds which also satisfy Formula X2:
Formula X2: 
With reference to Formula X2, A is oxygen or sulfur, and R1-R6 are independently selected from the group consisting of H, OH, halogen, aryl, arylamine, alkyl, alkene, substituted alkyl (such as alkylamine, alkylthio, haloalkyl, and substituted alkyls having two or more of such substituents), alkoxy, substituted alkoxy (such as alkoxyamine, alkoxythio, haloalkoxy, and alkoxy groups having two or more of such substituents) amino, ester, ether, nitro groups and O-linked and C-linked carbohydrates. Particular compounds satisfying Formula X2 have R1 and R6 selected from the group consisting of H, xe2x80x94OH, OR, whre R typically is lower alkyl, such as OCH3, and halogen. R2-R5 preferably are selected from the group consisting of side chains linked to the aromatic rings by a carbonyl or thiocarbonyl (i.e., Cxe2x95x90O or Cxe2x95x90S, respectively), a methylene group (i.e., CH2), oxygen, nitrogen or sulfur, and which further have a positively charged group on the terminal end of the linker. Such side chains are represented by Formula X3.
Formula X3: 
With reference to Formula X3, xe2x80x9cAxe2x80x9d is carbon, generally a methylene group, a carbonyl, amido, O, S, or N; xe2x80x9cnxe2x80x9d ranges from 1 to 10, preferably 2 to 8, and even more preferably from about 3 to 7 both branched chains or linear chains; and xe2x80x9cPxe2x80x9d is a group positively charged at physiological pH, such as amines, amidines, guanidines, cycloalkylamines, such as dicyclopropyl amine, or cycloalkylimines, such as pyrrolidine. xe2x80x9cnxe2x80x9d is selected to provide a chain length so that xe2x80x9cPxe2x80x9d preferably can interact with negatively charged groups, such as the propionate groups of heme. Compounds having superior biological activity against Plasmodium and Leishmania have been made using alkoxy amines, such as those represented by Formula X4.
Formula X4: 
With reference to Formula X4, xe2x80x9cnxe2x80x9d ranges from about 1 to 10, preferably 2 to 8, and even more preferably from about 3 to 7, including both branched chains and linear chains, and xe2x80x9cRxe2x80x9d typically is selected from the group of hydrogen and alkyl or cyclo-alkyl groups, preferably lower alkyl groups, such as ethyl groups. Moreover, the R groups of Formula X4 typically, but not necessarily, are the same or linked in a cyclic structure, such as pyrrolidine.
Specific examples of such compounds include 3,6-bis-N,N-diethylaminoxanthone, 3,6-bis-xcex2-(N,N-diethylamino)ethoxyxanthone, 3,6-bis-xcex3-(N,N-diethylamino)propoxyxanthone, 3,6-bis-xcex4-(N,N-diethylamino)butoxyxanthone, 3,6-bis-xcex5-(N,N-diethylamino)amyloxyxanthone, 3,6-bis-xcex6-(N,N-diethylamino)hexyloxyxanthone, 3,6-bis-xcex7-(N,N-diethylamino)heptyloxyxanthone, 3,6-bis-xcex8-(N,N-diethylamino)octyloxyxanthone, 3,6-bis-l-(N,N-diethylamino)nonyloxyxanthone, and 3,6-bis-xcexa-(N,N-diethylamino)decyloxyxanthone. 4,5 bis substituted amines and alkoxyamine analogs of these 3,6 bis-substituted compounds also are preferred compounds, including 4,5-bis-N,N-diethylaminoxanthone, 4,5-bis-xcex2-(N,N-diethylamino)ethoxyxanthone, 4,5-bis-xcex3-(N,N-diethylamino)propoxyxanthone, 4,5-his-xcex4-(N,N-diethylamino)butoxyxanthone, 4,5-bis-xcex5-(N,N-diethylamino)amyloxyxanthone, 4,5-bis-xcex6-(N,N-diethylamino)hexyloxyxanthone, 4,5-bis-xcex7-(N,N-diethylamino)heptyloxyxanthone, 4,5-bis-xcex8-(N,N-diethylamino)octyloxyxanthone, 4,5-bis-l-(N,N-diethylamino)nonyloxyxanthone, and 4,5-bis-xcexa-(N,N-diethylamino)decyloxyxanthone.
The disclosed invention also is directed to compositions comprising the compounds described above, i.e., compositions including compounds according to Formulae X, X1 and X2. These compositions are useful for the treatment of microbial diseases, such as malaria. These compositions may include materials conventionally used to make therapeutic compositions, and further may include additional therapeutics, particularly those useful for treating parasitic infections, such as malaria and leishmania. Examples, without limitation, of such therapeutics include chloroquine, antifolates, mefloquine, primaquine, cinchona alkaloids, such as quinine, sulfonamides, sulfones, tetracyclines, melarsoprol, nifurtimox, aminoacridines, aminoquinolines, sulfanolimides, pentamidine, stibogluconate, suramin, protease inhibitors, and mixtures thereof.
Also included in the present invention is a method of inhibiting the growth of a microbial pathogen. The method comprises providing a sufficient amount of a compound having Formulae X, X1 and/or X2, or composition comprising such compounds, and contacting the microbial pathogen with such compound(s) or composition(s). The present method is useful for inhibiting microbial growth in vivo and in vitro. In one aspect, the present invention provides a method for treating a patient having a microbial infection. xe2x80x9cPatientxe2x80x9d includes, without limitation, humans and animals, particularly economically important animals, such as livestock and avians, particularly poultry infected with protozoans, such as Eimeria. The method comprises administering to the patient a therapeutically effective amount of a compound or compounds, or composition comprising such compound or compounds, satisfying Formulae X, X1 and/or X2.
Another aspect of the present invention is the discovery that certain compounds having the xanthone ring structure depicted in Formulae X, X1 and/or X2 bind to, and inhibit the aggregation of, heme. A number of pathogens, including Plasmodium, a causative agent of malaria, degrade hemoglobin to obtain amino acids, and in so doing liberate toxic heme (Olliaro and Goldberg, 1995). To avoid the toxic effects of the liberated heme, these pathogens have evolved a mechanism for xe2x80x9caggregationxe2x80x9d of heme units to form hemozoin. (Pagola, Stephens, P. W., Bohle, D. S., Kosar, A. D., Madsen, S. K., Nature, xe2x80x9cThe Structure of Malaria Pigment Beta Haematin,xe2x80x9d 404:307-310 (2000). The compounds disclosed herein which are shown to inhibit heme aggregation may thus be used to block heme aggregation and therefore to treat infections caused by these pathogens. These heme complexing compounds may kill pathogens by preventing these organisms from gaining access to the host""s supply of heme iron, or by causing a build-up of toxic levels of heme in the organism. The compounds also may bind to heme of other metalloporphyrins and block one-electron transfer reactions.
Compounds which are disclosed herein to inhibit heme aggregation may be represented by the structure
Xxe2x80x94Yxe2x80x94Z 
where X is a group capable of interacting with the iron atom in heme (e.g., carbonyl, Nxe2x86x92O, Nxe2x80x94OH, SO2, and Sxe2x95x90O); Y is a substantially planar aromatic system capable of interacting with the porphyrin ring of heme, possibly through overlapping pixe2x80x94pi orbitals; and Z represents one or more groups capable of interacting with at least one carboxylate side group of heme. In preferred embodiments, these compounds are Formula X2 compounds.
The present invention also is directed to compositions useful for treating diseases, such as malaria, which are caused by pathogens that polymerize heme. The compositions include a compound according to Formula X2. Also included in the present invention is a method for inhibiting the growth of such a pathogen comprising providing a sufficient amount of a Formula X2 compound and contacting the pathogen with this compound. Such a method is applicable to inhibit pathogen growth in vivo and in vitro. In one aspect, the present invention provides a method for treating a patient having malaria, the method comprising administering to the patient a therapeutically effective amount of a compound according to Formula X2.
The invention also contemplates that Formula X, X1 and X2 compounds can be administered to patients in a pro-drug form. One example of a class of such prodrugs is correspondingly substituted benzophenones. These substituted benzophenones may react under physiological conditions to produce active compounds (i.e., the corresponding xanthone derivatives) satisfying Formulae X, X1 and/or X2 (Winter et al., 1996).
Related to the ability of Formula X, X1 and X2 compounds to bind to heme is the ability of these compounds to bind to a porphyrin. This porphyrin binding activity may be exploited in the development of treatments for porphyria. In addition, the binding between the Formula X, X1 and/or X2 compounds and heme/porphyrins may find applications in other contexts, such as laundry detergents (e.g., to enhance the ability of detergents to remove blood or grass stains) or in agricultural products that bind chlorophyll (a metalloporphyrin) and perturb plant growth.